Cleavage Site Specificities of Silkworm Alkaline Proteases

The cleavage site specificities of two alkaline proteases isolated from digestive juice of the silkworm larva, Bombyx mori, were analyzed using insulin A and B chains, α-lactalbumin, and egg white lysozyme. One, named P-IIc, could cut Arg-X and Lys-X peptide bonds and another, named P-IIIa, required rather hydrophobic amino acids not only at P1 but also at P4 sites. The effects of some protease inhibitors on P-IIIa could be explained by this requirement.     

Proteolytic enzymes from the mouse submaxillary gland: A partial sequence and demonstration of spontaneous cleavages

The mouse submaxillary proteases (A and D), whose isolation and properties were previously described by us, hydrolyze only arginyl bonds in proteins. The sequence of 40 residues from the amino terminus has been determined. Its structure shows a striking (>50%) homology with other enzymes of the serine group (e.g., trypsin, prothrombin, plasminogen, kallikrein). A single peptide labeled with diisopropylfluoro[³²P]phosphate had a composition virtually identical to that found in the above proteases, and one active site per molecule was confirmed. The enzymes are very susceptible to spontaneous fragmentation which leads to two cleavages. The first converts enzyme A to D with loss of a small peptide. The second can only be demonstrated after reduction since the fragments appear to be joined by a disulfide bridge. The two resulting fragments with molecular weights of 14,000 and 11,000, respectively, are inactive.     

Cleavage specificity of boar acrosin on polypeptide substrates, ribonuclease and insulin B-chain.

The cleavage specificity of boar acrosin is, like that of trypsin, strictly limited to the arginyl and lysyl bonds, as demonstrated for the oxidized B-chain of insulin. In addition, in this polypeptide substrate as well as in reduced and carboxymethylated ribonuclease, these peptide bonds are hydrolyzed by acrosin and trypsin with nearly identical velocities.     

Substrate specificity of tissue-type and urokinase-type plasminogen activators

Recent studies suggest that plasminogen activators not only hydrolyse a specific arginine-valine bond in plasminogen, but may also cleave other proteins such as fibronectin. We studied the substrate specificity, particularly the preference for arginyl over lysyl peptide bonds, of tissue-type plasminogen activator (t-PA) as well as of two-chain urokinase-type plasminogen activator (u-PA). The arginine/lysine preference was determined with three pairs of tripeptidyl-p-nitroanilide substrates having either arginine or lysine in the P1 position and varied from 5.2 to 14.1 for u-PA and from 55.6 to 99.8 for t-PA. It was concluded that both t-PA and u-PA preferred arginyl to lysyl peptide bonds. However, u-PA had a significantly lower arginine/lysine preference than t-PA, indicating that u-PA represents a less specific proteinase. This may point to functions of u-PA other than plasminogen activation, which involve cleavage of lysyl bonds.     

Unfolding rates of globular proteins determined by kinetics of proteolysis

A convenient method for the determination of unfolding rates of small globular proteins under physiological conditions was developed using digestion with proteases. The apparent first-order rate constants for digestion of lysozyme with thermolysin and with Pronase at pH 8 and 50 degrees C were shown to be saturated with increases of concentrations of these proteases. The maximum rate constants extrapolated were identical in digestions with two different proteases, and were found to be equal to the unfolding rate constant of lysozyme. Similarly, the unfolding rate constant of RNase A at pH 8 and 50 degrees C, and those of lysozyme, RNase A and beta-lactoglobulin at pH 8 and 40 degrees C, were determined by the digestion method. Thus, it was shown that digestion by proteases proceeds mainly via the unfolded state of proteins.     

The reactivity of the disulphide bonds of purified proteins in relationship to primary structure

1. With the aid of a coupled system involving glutathione reductase, the reaction of glutathione with the disulphide bonds of purified proteins has been studied. 2. Bovine serum albumin, conalbumin, lysozyme, trypsin inhibitors from egg white, lima bean and soya bean either did not react with glutathione or reacted only slightly. With these proteins reactivity was markedly increased by limited proteolysis. 3. Bovine and human gamma-globulins, fibrinogen and beta-lactoglobulin exhibited some reactivity (less than 15%) with glutathione and again this was increased by limited proteolysis. Pepsin, trypsin and chymotrypsin exhibited greater reactivity than the proteins previously mentioned. Di-isopropylphosphoryl-chymotrypsin exhibited less reactivity than chymotrypsin, suggesting that autolysis under the experimental conditions used contributed towards the reactivity of this protein. Proteolysis also increased the reactivity of these proteins. The three disulphide bonds of insulin were reduced by glutathione. 4. Above 35 degrees the disulphide bonds of serum albumin show a progressive increase in reactivity and at 55 degrees half of the bonds become accessible to glutathione. 5. From the results obtained with the proteins investigated, the conclusion reached is that the disulphide bonds of native proteins are structurally protected and do not react with glutathione under physiological conditions.     

Role of disulfide bonds in goose-type lysozyme

The role of the two disulfide bonds (Cys4-Cys60 and Cys18-Cys29) in the activity and stability of goose-type (G-type) lysozyme was investigated using ostrich egg-white lysozyme as a model. Each of the two disulfide bonds was deleted separately or simultaneously by substituting both Cys residues with either Ser or Ala. No remarkable differences in secondary structure or catalytic activity were observed between the wild-type and mutant proteins. However, thermal and guanidine hydrochloride unfolding experiments revealed that the stabilities of mutants lacking one or both of the disulfide bonds were significantly decreased relative to those of the wild-type. The destabilization energies of mutant proteins agreed well with those predicted from entropic effects in the denatured state. The effects of deleting each disulfide bond on protein stability were found to be approximately additive, indicating that the individual disulfide bonds contribute to the stability of G-type lysozyme in an independent manner. Under reducing conditions, the thermal stability of the wild-type was decreased to a level nearly equivalent to that of a Cys-free mutant (C4S/C18S/C29S/C60S) in which all Cys residues were replaced by Ser. Moreover, the optimum temperature of the catalytic activity for the Cys-free mutant was downshifted by about 20 degrees C as compared with that of the wild-type. These results indicate that the formation of the two disulfide bonds is not essential for the correct folding into the catalytically active conformation, but is crucial for the structural stability of G-type lysozyme.     

Evidence for mutual exclusion in the tryptic hydrolysis of two peptide bonds in maleylated trypsinogen

Treatment of completely maleylated trypsinogen with trypsin resulted in a mutual exclusion of hydrolysis of the two arginyl peptide bonds wherein one half of the molecules of the modified zymogen were cleaved at the Arg55-Leu56 bond and the other half of the molecules were cleaved at the Arg105- Val106 bond and no molecules were cleaved at both arginyl bonds.     

Characterization and peptidase specificity of lugworm (Arenicola cristata) protease C

1. Lugworm protease C further purified by benzamidine-affinity chromatography, exhibited peptidase specificity for arginyl and lysyl bonds. 2. Protease C consisted of a single polypeptide with a mol. wt of ca 23,000 as determined by SDS polyacrylamide gel electrophoresis, exhibited a u.v. absorption maximum at 280 with an (mg/ml) extinction coefficient of 0.93 and fluorescence spectra typical of a protein containing tryptophan, and had an amino acid composition similar to trypsins. 3. The Kms of the cleavages of the arginyl bond of oxidized insulin B chain and of the lysyl bond of the gly23-ala30 fragment were determined to be 0.72 and 0.96 mM; the corresponding kcats were 38 sec-1 and 1.5 sec-1. The Km and kcat for TAME were 0.042 mM, and 110 sec-1. 4. Lugworm protease C was confirmed to be a trypsin.     

The resistance to tryptic hydrolysis of peptide bonds adjacent to N epsilon,N-dimethyllysyl residues

A peptide from sperm whale myoglobin, residues 132-153, and a chromogenic substrate, H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide diacetate, were selected to investigate the susceptibility of peptide bonds adjacent to N epsilon,N-dimethyllysyl residues to tryptic hydrolysis. The peptides were exhaustively methylated using formaldehyde and sodium cyanoborohydride (N. Jentoft and D. G. Dearborn (1979) J. Biol. Chem. 254, 4359-4365). Unmodified and methylated peptides were digested with trypsin or submaxillary protease, an enzyme that catalyzes the hydrolysis of only arginyl bonds in proteins. Trypsin catalyzed the hydrolysis of the methylated apomyoglobin peptide only at the single arginyl residue and not at any of the four N epsilon,N-dimethyllysyl residues. Trypsin also failed to catalyze the hydrolysis of reductively methylated H-D-valyl-L-leucyl-L-lysyl-p-nitroanilide. Even a 17-fold molar excess of the methylated substrate did not appear to alter the rate of tryptic hydrolysis of the unmodified substrate. These results are discussed with regard to the interactions of substrates within the specificity site of trypsin.     

Enabling Low Cost Biopharmaceuticals: A Systematic Approach to Delete Proteases from a Well-Known Protein Production Host Trichoderma reesei

The filamentous fungus Trichoderma reesei has tremendous capability to secrete proteins. Therefore, it would be an excellent host for producing high levels of therapeutic proteins at low cost. Developing a filamentous fungus to produce sensitive therapeutic proteins requires that protease secretion is drastically reduced. We have identified 13 major secreted proteases that are related to degradation of therapeutic antibodies, interferon alpha 2b, and insulin like growth factor. The major proteases observed were aspartic, glutamic, subtilisin-like, and trypsin-like proteases. The seven most problematic proteases were sequentially removed from a strain to develop it for producing therapeutic proteins. After this the protease activity in the supernatant was dramatically reduced down to 4% of the original level based upon a casein substrate. When antibody was incubated in the six protease deletion strain supernatant, the heavy chain remained fully intact and no degradation products were observed. Interferon alpha 2b and insulin like growth factor were less stable in the same supernatant, but full length proteins remained when incubated overnight, in contrast to the original strain. As additional benefits, the multiple protease deletions have led to faster strain growth and higher levels of total protein in the culture supernatant.     

Selective activation of the 20 S proteasome (multicatalytic proteinase complex) by histone h3.

Two distinct activities cleaving bonds after hydrophobic amino acids have been identified in the bovine pituitary 20 S proteasome. One, expressed by the X subunit, that cleaves bonds after aromatic and branched chain amino acids was designated as chymotrypsin-like (ChT-L).(1) The second, expressed by the Y subunit, that cleaves bonds after acidic amino acids was designated as peptidylglutamyl-peptide hydrolyzing (PGPH) but also cleaves bonds after branched chain amino acids. Low micromolar concentrations of the arginine-rich histone H3 (H3) are shown to induce changes in the specificity of the proteasome by selectively activating cleavages after branched chain and acidic amino acids while inhibiting cleavage of peptidyl-arylamide bonds in synthetic substrates. H3 activates 15-fold cleavage after leucine but not phenylalanine residues in model synthetic substrates. The activation is associated with a decrease in K(m) and an increase in V(max), suggesting positive allosteric activation. H3 activates more than 60-fold degradation of the oxidized B-chain of insulin, by cleaving mainly bonds after acidic and branched chain amino acids, and accelerates the degradation of casein and lysozyme, the latter in the presence of dithiothreitol. The degradation of lysozyme in the presence of H3 generates fragments that differ from those in its absence, indicating H3-induced specificity changes. H3 inhibits cleavage of the Trp3-Ser4 and Tyr5-Gly6 bonds in gonadotropin releasing hormone, bonds cleaved by the ChT-L activity in the absence of H3. The results suggest H3-selective activation of the Y subunit and specificity changes that could potentially affect proteasomal function in the nuclear compartment.     

Basic proline-rich proteins from human parotid saliva: relationships of the covalent structures of ten proteins from a single individual.

Eleven basic proline-rich proteins were purified from the parotid saliva of a single individual. The complete amino acid sequences of six of these were determined by conventional protein sequence methodology, bringing to nine the number of known primary structures of nonglycosylated basic proline-rich proteins from the same individual. The partial sequence of one additional protein is also reported. All of the basic proline-rich proteins studied contain segments with identical or very similar sequences, but with two possible exceptions, none of the proteins is derived from another secreted proline-rich protein. The amino acid sequences of nine nonglycosylated basic proline-rich proteins were compared with primary structures deduced from published nucleotide sequences of DNA coding for human parotid proline-rich proteins. The sequences align well, in general, but differences also exist pointing to the complexity of the genetics of these proteins. Seven secretory basic proline-rich proteins appear to be formed from three larger precursors by selective posttranslational proteolyses of arginyl bonds. One of the basic proline-rich proteins appears to derive from human acidic proline-rich proteins. The remaining two proteins studied do not conform to any DNA structure as yet reported. Two of the basic proline-rich proteins studied are phosphoproteins and exhibit abilities to inhibit hydroxyapatite formation in vitro.     

Degradation of proteins by enzymes exuded by Allium porrum roots–A potentially important strategy for acquiring organic nitrogen by plants

Nitrogen is one of the crucial elements that regulate plant growth and development. It is well-established that plants can acquire nitrogen from soil in the form of low-molecular-mass compounds, namely nitrate and ammonium, but also as amino acids. Nevertheless, nitrogen in the soil occurs mainly as proteins or proteins complexed with other organic compounds. Proteins are believed not to be available to plants. However, there is increasing evidence to suggest that plants can actively participate in proteolysis by exudation of proteases by roots and can obtain nitrogen from digested proteins. To gain insight into the process of organic nitrogen acquisition from proteins by leek roots (Allium porrum L. cv. Bartek), casein, bovine serum albumin and oxidized B-chain of insulin were used; their degradation products, after exposure to plant culture medium, were studied using liquid chromatography–mass spectrometry (LC–MS). Casein was degraded to a great extent, but the level of degradation of bovine serum albumin and the B-chain of insulin was lower. Proteases exuded by roots cleaved proteins, releasing low-molecular-mass peptides that can be taken up by roots. Various peptide fragments produced by digestion of the oxidized B-chain of insulin suggested that endopeptidase, but also exopeptidase activity was present. After identification, proteases were similar to cysteine protease from Arabidopsis thaliana. In conclusion, proteases exuded by roots may have great potential in the plant nitrogen nutrition.     

Aggregate formation and the structure of the aggregates of disulfide-reduced proteins

Aggregate formation and the structure of the aggregates of disulfide-reduced proteins were investigated using -lactalbumin and lysozyme as model proteins. First, reducing conditions were adjusted so that only one of the four disulfide bonds present in each native protein was cleaved. These three-disulfide (3SS) proteins are known to adopt almost native conformations, yet formed precipitates with a basic peptide, lactoferricin, and heparin and heparin fragment, respectively, at concentrations at which native proteins mixed with these compounds remained clear. The 3SS-lysozyme also formed precipitates in the absence of these ligands. Thus, subtle structural changes could lead to aggregation. Electron microscopy revealed fibrillar structures in the aggregates of extensively reduced proteins in the absence of ligands but not in their presence, which shows that the reduction of disulfide bonds suffices for fibril formation and that ligands inhibit fibril formation.     

Proteolysis approach without chemical modification for a simple and rapid analysis of disulfide bonds using thermostable protease‐immobilized microreactors

Disulfide bonds in proteins are important not only for the conformational stability of the protein but also for the regulation of oxidation–reduction in signal transduction. The conventional method for the assignment of disulfide bond by chemical cleavage and/or proteolysis is a time‐consuming multi‐step procedure. In this study, we report a simple and rapid analysis of disulfide bond from protein digests that were prepared by the thermostable protease‐immobilized microreactors. The feasibility and performance of this approach were evaluated by digesting lysozyme and BSA at several temperatures. The proteins which stabilize their conformations by disulfide bonds were thermally denatured during proteolysis and were efficiently digested by the immobilized protease but not by free protease. The digests were directly analyzed by ESI‐TOF MS without any purification or concentration step. All four disulfide bonds on lysozyme and 10 of 17 on BSA were assigned from the digests by the trypsin‐immobilized microreactor at 50°C. The procedure for proteolysis and the assignment were achieved within 2 h without any reduction and alkylation procedure. From the present results, the proteolysis approach by the thermostable protease‐immobilized microreactor provides a strategy for the high‐throughput analysis of disulfide bond in proteomics.     

Effect of disulfide bonds on lysozyme dynamics

The effect of destruction of disulfide bonds on the dynamics of proteins was studied by an example of lysozyme by the methods of molecular dynamics. In lysozyme, in the absence of disulfide bonds, the characteristic times of motions of secondary structure devices increased 3-7 times, whereas the amplitudes of fluctuations of secondary structure devices practically did not vary. In the absence of S-S-bonds, the volume of the molecule decreased approximately by 2%, primarily due to a "cleft" between the major and the small domains of lysozyme. Thus, disulfide bonds not only "glue" the secondary structure devices of the protein but also play a role of "rods", maintaining a certain free volume of the molecule necessary for the realization of its functions.     

Involvement of Amino-Acid Side Chains of Membrane Proteins in the Binding of Glutathione to Pig Cerebral Cortical Membranes

Glutathione (GSH), a general antioxidant and detoxifying compound, is the most abundant thiol-containing peptide in the central nervous system. It has been earlier shown to regulate the functions of glutamate receptors and to possess specific binding sites in both neurons and glial cells. The possible involvement of disulfide bonds, cysteinyl, arginyl, lysyl, glutamyl, and aspartyl residues in the binding of tritiated GSH to specific sites in pig cerebral cortical synaptic membranes was now studied after covalent modification of membrane proteins. Treatment of synaptic membranes with the thiol-modifying reagents 5,5-dithio-bis(2-nitrobenzoate) (DTNB) and 4,4-dithiodipyridine (DDP) dramatically enhanced the binding of [³H]GSH in a dose-dependent manner. Dithiothreitol (DTT) alone reduced the binding, but pretreatment of the membranes with DTT potentiated the enhancing effect of DTNB. On the other hand, when the modification with DTNB was followed by treatment with DTT, the enhancement by DTNB was completely reversed. N-ethylmaleimide, a thiol alkylating agent, and phenylisothiocyanate, a thiol- and amino-group modifying compound, reduced the binding, and their effects were additive. The guanidino-modifying agent phenylglyoxal reduced the binding but the carboxyl-modifying reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide had no significant effect. The results indicate that cysteinyl side chains and disulfide bonds are essential in the binding of GSH to membrane proteins and that arginyl and lysyl side chains may also be directly involved in this process.     

Evidence for difference in the roles of two cysteine residues involved in disulfide bond formation in the folding of human lysozyme

Human lysozyme is made up of 130 amino acid residues and has four disulfide bonds at Cys6-Cys128, Cys30-Cys116, Cys65-Cys81, and Cys77-Cys95. Our previous results using the Saccharomyces cerevisiae secretion system indicate that the individual disulfide bonds of human lysozyme have different functions in the correct in vivo folding and enzymatic activity of the protein (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967). In this paper, we report the results of experiments that were focused on the roles of Cys65 and Cys81 in the folding of human lysozyme protein in yeast. A mutant protein (C81A), in which Cys81 was replaced with Ala, had almost the same enzymatic activity and conformation as those of the native enzyme. On the other hand, another mutant (C65A), in which Cys65 was replaced with Ala, was not found to fold correctly. These results indicate that Cys81 is not a requisite for both correct folding and activity, whereas Cys65 is indispensable. The mutant protein C81A is seen to contain a new, non-native disulfide bond at Cys65-Cys77. The possible occurrence of disulfide bond interchange during our mapping experiments cannot be ruled out by the experimental techniques presently available, but characterization of other mutant proteins and computer analysis suggest that the intramolecular exchange of disulfide bonds is present in the folding pathway of human lysozyme in vivo.     

Rates of pinocytic capture of simple proteins by rat yolk sacs incubated in vitro.

The rates of pinocytic uptake of a number of small 125I-labelled simple proteins (insulin, ribonuclease A and lysozyme) by rat yolk sacs incubated in vitro were determined both before and after treating these proteins with reagents that are known to increase the rate of capture of 125I-labelled bovine serum albumin. Uptake of the untreated forms of all three proteins was extremely rapid, indicating that adsorptive pinocytosis is the principal mechanism by which yolk-sac cells capture these simple proteins, but these rates show no simple correlation with molecular charge. In contrast with albumin, the rates of uptake of treated proteins were either unchanged or lower than that of the corresponding untreated protein preparations; polymeric forms of 125I-labelled lysozyme larger than dimers were ingested at rates significantly lower than that of the monomer.